Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity

Abstract

Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the successful development of assisted reproduction procedures. The aim of this study was to assess the mitochondrial membrane potential, lipid peroxidation, and DNA integrity of equine sperm subjected to freezing, vitrification, and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses were subjected to programmable freezing, vitrification, and freeze-drying. After thawing or rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial membrane potential (ΔΨM), lipid peroxidation (LPO), and DNA fragmentation index (DFI) of the spermatozoa were assessed by flow cytometry using the DiOC6 (3), C11-Bodipy 581/591, and propidium iodide (PI) fluorescent dyes. The statistical analysis was conducted via generalized linear models, mean comparisons via the Duncan test, and a principal component analysis. A higher rate of spermatozoa with a high ΔΨM was found for freeze-drying (40.26 ± 7.79%) compared with freezing (21.82 ± 5.38%) and vitrification (5.32 ± 1.17%) (P < .05). Likewise, a higher rate of nonperoxidized viable spermatozoa (Bodipy-/PI-) was found for freeze-drying (35.98 ± 7.01%) in relation to frozen (10.34 ± 2.69%) and vitrified (7.07 ± 2.00%) sperm (P < .05). The DFI of vitrified spermatozoa (0.12 ± 0.04%) was higher when compared with the frozen (0.03 ± 0.01%) and freeze-dried (0.02 ± 0.01%) samples (P < .05). The researchers conclude that vitrification generates greater sperm alterations than freeze-drying and freezing, whereas freeze-drying produces lower LPO and higher ΔΨM for equine spermatozoa.